Although many molecular details of the signaling pathways in lymphocytes have been elucidated with exquisite detail, less is known about the equally important aspects of how immune cells migrate, locate one another and transmit signals within their native tissues. There is thus a pressing need for techniques allowing real-time observation of single cells and molecules in lymphoid organs. We have pioneered the use of two- photon microscopy to track motility and cell/cell interactions of dendritic cells, T cells and B cells in isolated lymph nodes and in living mice. This proposal for functional imaging has three inter-related Aims. First, we will examine basic motility properties of temperature dependence, effects of tissue hypoxia and regional variations in the lymph node under basal conditions and at the start of an immune response. The control of lymphocyte trafficking by S1P1 receptors will be probed using specific pharmacological tools and by directly imaging the egress step to determine mechanisms of lymphocyte sequestration. We introduce quantum dots as a novel approach to track and modulate dendritic cells. Second, we will focus on immunoregulatory and effector interactions among cells in the lymph node. We introduce regulatory T (Treg) cells and natural killer (NK) cells as new and relevant cells for immuno-imaging, and extend our approach to include human immune cells reconstituted or transferred into mice. Third, we propose to use [Ca2+]i imaging, K+ channel and PI 3-kinase knockout mice, and specific pharmacologicaltools to investigate molecular signaling pathways that regulate motility and antigen responses in the lymph node. We anticipate that the advances in technology development and new information gained through this project will provide the means to investigate autoimmune diseases and further the mechanistic understanding of clinically used immunosuppressive agents.